1. Field of the Invention
Diagnostic procedures based on the enzymatic determination of substances in biological fluids are well-known. In the enzymatic determination of alcohol or other dehydrogenase/oxidase substrates in body fluids, enzyme assay systems comprising a dehydrogenase and a cofactor for the enzyme (usually NAD+) are typically employed to oxidize the substrate and simultaneously reduce the cofactor to a readily assayable product.
In the case of an alcohol, selective oxidation by such enzyme systems results in the production of the corresponding free aldehyde or ketone. The presence of these oxidation products are very detrimental to the assay, as firstly, they tend to inactivate the enzymes employed in the system, and, secondly, they unfavorably shift the dehydrogenation reaction equilibrium so that oxidation of the substrate does not tend to go to completion. Since assay results under these conditions are unreliable, enzyme assay systems for the determination of alcohol now typically require dilution of the substrate and/or include complexing or trapping agents for the oxidation products which in essence remove free aldehyde or ketone from the reaction.
2. Description of Related Art
A variety of agents for trapping alcohol oxidation products, especially acetaldehyde produced by enzymatic dehydrogenation of ethanol, have been proposed. In commercial applications, the class of agents generally employed comprises primary amines, which react with free aldehyde to form the corresponding imine. Since the product imine is less reactive than the precursor aldehyde or ketone, enzyme deactivation is reduced, and the dehydrogenation reaction equilibrium is favorably shifted. Even with the primary amine trapping agents, however, the alcohol dehydrogenation typically still does not go substantially to completion, with often less than about 20% of the alcohol converted. Further, assay systems employing a primary amine as trapping agent for alcohol oxidation products are generally only useful for assaying fluids having a low alcohol content and a correspondingly low production of aldehyde or ketone oxidation product. In general, the usefulness of these enzyme assay systems is limited to samples producing less than about 5 mM acetaldehyde, more typically less than about 1 mM acetaldehyde, as the known primary amine trapping agents cannot effectively remove larger amounts from the assay system. For instance, acetaldehyde in concentration greater than 1 mM inhibits the enzyme alcohol dehydrogenase (ADH). Since analysis of body fluid samples potentially containing in excess of about 150 mM alcohol is routine, such samples must be diluted prior to assay to reduce target concentrations to an assayable level. For example, in situations where 21 mM blood ethanol is the legal definition of intoxication, and the sample is to be tested for this concentration, the sample must be diluted sufficiently to reduce the alcohol concentration thereof to within the assay range; generally to less than about 5 mM, depending upon the particular assay system. This must be done very carefully to ensure an accurate determination.
An example of known systems is the Abbott TDX Analyzer. This device requires extensive dilution of the sample to be tested so that only 1 mM of acetaldehyde is produced since otherwise the undesirable results discussed above will take place. The Abbott Analyzer uses an acetaldehyde trap, 2-amino-2-methyl-1,3-propanediol ("Tris amino" or "Tris") as a buffer.
Another illustration of this type of assay is illustrated by U.S. Pat. No. 3,926,736 to Buccolo (Calbiochem), 1975. The patent discloses the use of Tris buffer as a trapping agent. The samples to be analyzed are diluted so that only about 0.5 mM acetaldehyde is produced.
U.S. Pat. No. 4,481,292 to Raymond (The Coca-Cola Company), 1984, also shows the use of Tris buffer as trapping agent for high concentrations of acetaldehyde. The reactor is adapted for and the process is a continuous flow type.
The patent describes the difficulties encountered in a practical enzyme-catalyzed process for converting ethanol to acetaldehyde, including equilibrium considerations which favor acetaldehyde conversion to alcohol. Although concentrations of acetaldehyde on the order of 85 mM are disclosed, it is essential in accordance with the process (and apparatus) that the alcohol dehydrogenase (ADH) be separated and confined during the reaction sequence. The ADH is separated by a semi-permeable membrane from the acetaldehyde and the system permits the passage of the starting materials as well as the products formed so that a liquid flow away from the enzyme takes place on a continuous basis. Thus, the enzyme is never exposed to high concentrations of acetaldehyde. Even under such conditions, the yield of acetaldehyde is quite limited, the conversion of the alcohol being about 18%. The efficiency of the amine buffer system is not a feature of the process, and "Tris" is not capable by itself of reacting with and removing such high concentrations of acetaldehyde.
The process and the system of the present invention does not call for a flow system, twice the amount of acetaldehyde can be trapped and 100% of the alcohol can be converted to acetaldehyde.
The system of the invention can be embodied and used as a dry film rather than a large continuous flow reactor. Other differences between the device of the invention and the prior art will become apparent in the description of the invention as presented herein.
Owing to the inability of these known systems to assay for routinely encountered concentrations of alcohols without sample dilution, the assay is of necessity a liquid assay with the reagents supplied in liquid form, usually as a kit. Sigma Chemical, for example, markets the following individual liquid reagents for the enzymatic determination of ethyl alcohol in blood samples: NAD-ADH; ethanol standard solution; hydrazine; glycine buffer reagent; trichloroacetic acid solution. These liquid reagents are bulky, and must be carefully combined in the proper amounts for each assay, refrigerated during storage, and protected against contamination and spillage.
Bostick and Overton (Biotechnol. Bioengin. 22:2383-92, 1980) also describe the addition of hydrazine to an enzymatic alcohol test, which increases the effectiveness of the measuring range to 3 mM, and the test format is liquid.
In Biochem. J., 104 p. 165 (1967), Dickinson and Dalziel, The Specificities and Configurations of Ternary Complexes of Yeast and Liver Alcohol Dehydrogenases, discuss the use of Tris as an acetaldehyde trapping agent. The authors note that Tris is an unsuitable buffer for equilibrium or initial-rate measurements with some carbonyl compounds. The authors state, however, that Tris does not afford a convenient buffer for the enzymatic estimation of small amounts of ethanol. Additional differences over that art will become apparent from the detailed discussions which follow.
It is accordingly desirable and there is a need to provide an assay in a fluid for the enzymatic determination of alcohol concentration, which is capable of reliably converting substantially 100% of the substrate, trapping high concentrations of ketone or aldehyde (in excess of 5 mM, e.g. in excess of 150 mM), does not deactivate enzyme reagents, is useful for determining high alcohol concentrations without sample dilution (e.g. in the range of 20 to 150 mM, or higher), which can be provided in easily useable and storable form such as a dry film format, and which is highly reliable in use, even for inexperienced assayers.